Ebola virus (EBOV) is a member of the filovirus family that causes severe viral hemorrhagic fever (VHF). Although infrequent, epidemics of EBOV can cause high mortality rates of up to 90%; the subsequent medical and social upheaval can be widespread and severe, as seen in the recent outbreak in Western Africa which has already caused more than 10,000 fatalities in a region with insubstantial medical services. Although some success using monoclonal and polyclonal antibodies has been reported, these expensive treatments are not widely available to poorer regions of Africa without substantial assistance from America and Europe; additionally, it would be beneficial to have the ability to stockpile treatments in case of future outbreaks or bioterrorism. This option is not easily achieved with antibody therapies. A pressing need for small-molecule therapeutics is thus quite evident. The experimental drugs brincidofovir, favipiravir, and BCX 4430 have shown promise in vitro, but no clinical trials have proven their effectiveness in vivo. To address this critical unmet medical need, new small-molecule therapeutics/prophylactics are necessary to avert the risk of future epidemics. Using a pseudotype virus that mimics the viral entry process of EBOV, we have identified a novel set of small molecule EBOV entry inhibitors that has been validated in assays of infectious EBOV in vitro. Based on a beta-lactam central core, these compounds are readily modified, are drug-like, and represent an excellent starting point for medicinal chemistry optimization. By synthesizing new analogs of the hit compound, MBX 2806, we will generate structure-activity relationships to better understand the chemical features that lead to potent anti-EBOV activity and low cytotoxicity, and ultimately produce potent, selective inhibitors of infectious EBOV that display drug-like characteristics. The current proposal will use medicinal chemistry to optimize MBX 2806 using three aims: 1) We will synthesize novel analogs of MBX 2806 and assay the antiviral activity in a pseudotype assay of EBOV infection. 2) We will validate the results of the pseudotype assay using infectious EBOV under BSL4 conditions. 3) We will measure in vitro ADME predictors to improve the overall drug-likeness of the scaffold. Using an iterative process of compound design, synthesis, and biological assay, we will synthesize optimized compounds that are potent, selective, and have drug-like properties suitable for further development as therapeutics and/or prophylactics for EBOV infection.